Study On Rapid Determination Of Sensitivity Of Ehinochloa Spp. And Alopecurus Aequalis To Some Frequently Used Herbicides

In order to select herbicides more precisely to control weeds in crop fields,a rapid,highly efficient and accurate method to determine the sensitivity of weeds to herbicides before spraying is of great importance for reducing herbicide use and delaying the evolving of herbicide resistance.In this paper,the RISQ(Resistance In-Season Quick)technology for determining sensitivity of Echinochloa spp.to herbicides commonly used in paddy fields(e.g.penoxsulam,oxazolamide,quinclorac)and Alopecurus aequalis to frequently used herbicide in wheat fields(e.g.methylsulfuron-methyl,imidazolam)were established.Meanwhile,a dCAPS(derived cleaved amplified polymorphic sequences)based technique was established for detecting the resistant individuals containing the 11781L mutation in ACCase and/or P197T and T574L mutations in the ALS of A.aequalis,respectively,whose accuracy and practicality were also confirmed.The main findings are described as follows:Six biotypes of Echinochloa spp.(JNBX-1?JHLS-1?AXX-8 and AXXZ-6 for E.crusg alli,JTJY-1 for E.glabrescens,and JNX-S for E.crusgalli var.zelayensis)to penoxsulam were screened by whole-plant bioassay.Using the same ways,Six biotypes of Echinochloa spp.(JNBX-1?AXXZ-2?AXX-8 and AXXZ-6 for crusgalli,JTJY-1 for E.glabrescens,and JNX-S for E.crusgalli var.zelayensis)to metamifop were also screened.Six biotypes of Echinochloa spp.(JCW-R?SSX-R and JNX-S for E.crusgalli var.zelayensis,JNBX-1 for E.crusgalli,and JTJY-1 for E.glabrescens)to quinclorac were also sreened by whole-plant bioassay.The RISQ technology was developed based on these biotypes of E.spp.mentioned above.Different sensitivity discriminating concentrations were obtained,which are 0.3?mol/L for E.crusgalli to penoxsulam,0.6?mol/L for E.crusgalli to metamifop and 2.4?mol/L for E.crusgalli var.zelayensis,respectively.Six biotypes of A.aequalis with different sensitivities to methylsulfuron-methyl(JNXW-1,JNJN-1,JYTG-1,JTJY-1,JGYS-1 and JHHZ-1)were screened by whole-plant bioassays.The RISQ detection technology was obtained that the sensitivity discriminating concentrations of methylsulfuron-methyl was 0.6 ?mol/L.Also,six biotypes of A.aequalis were sreened by whole-plant bioassays,including JNXW-1,JLGY-9,JYTG-1,JCWJ-3,JTJY-1,and JHHZ-1 to develop RISQ technique.It was also found that the sensitivity discriminating concentrations of pyroxsulam was 0.4 ?mol/L.Six populations(JNXW-1,JHHZ-1,JYTG-1,JCWJ-3,JTJY-1,JGYS-1)with different sensitivity levels were screened out in eight populations by whole-plant bioassays.Established RISQ detection technology and obtained the sensitivity discriminating concentrations of fenoxaprop-P-ethy 0.16?mol/L.Methods based on dCAPS to detect I1781L mutation in ACCase,P197T and/or T574L mutations in ALS of of fenoxaprop resistant and methylsulfuron-methyl resistant A.aequalis were developed,respectively.The results showed that when the PCR fragments targeting I1781L mutation in ACCase from resistant and sensitive individuals were digested by BamHI,two bands(271 bp and 49bp,respectively,in which the 49bp band could not be visualized on 3%agrose gel by electrophoresis)would be produced for the sentive indiduvals,while a 320bp band could be visualized on 3%argose gel for the resistant individuals,as the amplified fragment could not be digested by BamHI.For detectiong the P197T mutation in ALS,two bands(347 bp and 47 bp,respectively,in which the 47 bp band could not be visualized)could be produced for the mesosulfuron-methyl resistant individuals when digested by BamHI,while for the sensitive individuals,only one band(394 bp)could be visualized.For detecting the P574T mutation in ALS,two bands(325 bp and 45 bp,respectively,in which the 45 bp is not visualized)were produced for the sensitive individuals when digested by Hpa?,while only one band(370 bp)could be visualized for the mesosulfuron-methyl resistant individuals.To confirm the availibility of the dCAPs based methods,the results from dCAPS and PCR product sequencing were compared and the accuracy and reliability of the developed dCAPS based methods were validated.In order to further verify the practicability of the developed dCAPS based detecting methods,50 randomly selected individuals from mesosulfuron-methyl resistant and fenoxaprop resistant biotypes of A.aequalis were tested for detecting the different mutation,respectively.The results showed that in JYTG-1 and JGYS-1(resistant to fenoxaprop),the frequency of resistant individuals were 72%and 96%,respectively.And no P197T and T574L mutations were detected in JCWJ-3(resistant to mesosulfuron-methyl),while in JGYS-1,both P197T and T574L mututions were detected with frequency of 8%and 84%,respectively.From the results,it could be inferred that in JCWJ-3,there may be unknown mutations in ALS that could confer mesosulfuron-methyl resistance to A.aequalis,or non-target based resistance would exist.In JGYS-1,T574L mutation was a dominant mutation that could cause mesosulfruon-methyl resistance.Also,in all the resistant individual detected,only homozygous mutations were found.