Resistance And Target Mechanism Of Cyperus Difformis To Acetohydroxyacid Synthase Inhibitors

Cyperus difformis L.is an annual weed of sedge family,which distributes in Northeast,North,Central,East,Ningxia,Gansu and other areas of China,mainly harming in rice.AHAS inhibitors can effectively control the C.difformis in paddy field,but its single action site and continuous application for many years has lead to serious resistance.In this study,38 populations were collected from paddy fields in 7 provinces of Hubei,Hunan,Jiangxi,Jiangsu,Anhui,Liaoning and Heilongjiang.The objectives of this research were:?1?confirm the distribution of resistance to pyrazosulfuron-ethyl in C.difformis populations collected from paddy fields in 7 provinces of China;?2?sequence the complete AHAS gene and identify the mutation sites of AHAS in different populations;?3?detect the sensitivity difference of resistant C.difformis based on the AHAS in vitro activity in the presence of four AHAS inhibitors;and?4?determine the cross-resistance patterns to four AHAS inhibitors conferred by different AHAS mutations.The main results are listed below:?1?The result of single dose screening of pyrazosulfuron-ethyl showed that among 38 C.difformis populations,16 of which were survived under the recommended dose of 30 g a.i.ha^-1 of pyrazosulfuron-ethyl,with a survival rate of 25%-100%.It was preliminarily determined that these populations had developed resistance to pyrazosulfuron-ethyl.?2?The complete AHAS sequence of C.difformis was cloned by Genome Walking and other skills.The results showed that the intron was between two exons in complete AHAS sequence of C.difformis.The intron was 1228-1233 bp and the exons were 498 and 1428 bp,respectively.?3?Three pairs of primers were designed to amplify eight target-sites and to compare the differences between the resistant and susceptible populations.The results showed that comparing with the susceptible populations,16 resistant populations had mutations in the conservative region of AHAS.The mutation types were Pro-197 Arg/Ser/Leu,Asp-376-Glu?Glu?,Trp-574-Leu.There were multiple mutation types in some populations,such as Pro-197-Arg and Asp-376-Glu in YX12-14,Pro-197-Arg and Trp-574-Leu in YX12-15,Pro-197-Ser/Arg/Leu in YX15-22,Pro-197-Ser and Trp-574-Leu in YX15-32.?4?Through the extraction of AHAS with different mutation types,the difference of inhibition level of in vitro AHAS activity by four herbicides was detected.The results showed that the I₅₀ values of susceptible population YX15-30 were 0.06,0.01,3.29 and 0.04?M under the treatment of pyrazosulfuron-ethyl,penoxsulam,imazapic and bispyribac-sodium.Compared with the susceptible population,the resistance index?RI?of YX12-10?Asp-376-Glu?were 91.6,34.0,4.3 and 8.0;the RI of YX15-22?Pro-197-Ser/Arg/Leu?were 310.0,10.0,1.2 and 1.3;and the RI of YX15-38?Trp-574-Leu?were 4462.0,831.0,260.7 and 52.3,respectively.?5?The cross-resistance levels of different mutated populations to AHAS inhibitors were detected by whole plant bioassays and the result showed that the GR₅₀ values of susceptible population YX15-30were 1.10,3.10,3.31 and 6.53 g a.i.ha^-1,under the treatment of pyrazosulfuron-ethyl,penoxsulam,imazapic and bispyribac-sodium.Compared with the susceptible population,the RI of YX12-10?Asp-376-Glu?were 328.3,16.3,6.6 and 5.8;the RI of YX15-22?Pro-197-Ser/Arg/Leu?were 827.4,6.6,1.0 and 1.0;and the RI of YX15-38?Trp-574-Leu?were 3321.9,382.4,1293.8 and 154.33,respectively.In summary,it was the first time to clone the complete AHAS sequence of C.difformis,and identify five mutation types of AHAS gene in resistant population.Through the detection of target enzyme,it was clear that the sensitivity difference of different populations to AHAS inhibitor caused by different mutations of target gene,and the level of cross-resistance of different mutation types to AHAS inhibitor.