Resistance To Fenoxaprop-P-ethyl And Mesosulfuron-methyl In Shortawn Foxtail (Alopecurus Aequalis) From Wheat Fields

Shortawn foxtail?Alopecurus aequalis?is a common Gramineae weed widely distributed in east,south-central and southwest China and parts of the Yellow River basin.It is a competitive and troublesome weed in the middle and lower reaches of Yangtze river,mainly infesting in summer harvesting crops such as wheat?Triticum aestivum L.?and oilseed rape?Brassica napus L.?.Fenoxaprop-P-ethyl,which targets in acetyl-CoA carboxylase?ACCase?,is the most common herbicide in controlling grass weeds in wheat field of China.In some regions of Anhui and Jiangsu province,however,fenoxaprop-P-ethyl could no longer control shortawn foxtail effectively or even failed after repeated use.Since then,mesosulfuron-methyl,an acetolactate synthase?ALS?-inhibiting herbicide,become an important alternative choice for the control of fenoxaprop-P-ethyl-resistant weeds.Unfortunately,shortawn foxtail also evolved resistance to mesosulfuron-methyl rapidly,and some populations show multiple resistance to these two dissimilar herbicides,which become a severe threat in wheat production.This study mainly concerns in fenoxaprop-P-ethyl and mesosulfuron-methyl resistance in shortawn foxtail,and researches about resistance distribution,resistance levels,target and non-target resistance mechanism,cross resistance and fitness were conducted in order to use herbicide rationally,delay resistance evolution and manage the multiple resistant shortawn foxtail.The main results are showed below:1.Seventy-seven shortawn foxtail populations were collected from Anhui,Jiangsu,Shandong and Henan province,and were treated with fenoxaprop-P-ethyl(62.1 g a.i.ha^-1)and mesosulfuron-methyl(9 g a.i.ha^-1)at the recommended field rate respectively.The results showed that 37 and 34 populations were resistant to fenoxaprop-P-ethyl and mesosulfuron-methyl respectively,among which,27 populations displayed multiple resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl.The proportion of the resistant plant varied for different populations,which indicated that they were at different evolution stages.Whole-plant dose-response experiments were conducted to determine the resistance levels of shortawn foxtail field populations.Most populations showed high-level resistance to fenoxaprop-P-ethyl and/or mesosulfuron-methyl,with resistance index?RI?6.2¹78.6 and7.6¹39.5 respectively.2.As the cytochrome P450 inhibitor,piperonyl butoxide?PBO?and malathion were used in combination with fenoxaprop-P-ethyl and mesosulfuron-methyl respectively to evaluate the synergistic effect.The use of PBO(4200 g a.i.ha^-1)in combination with fenoxaprop-P-ethyl didn't have a significant effect on HNXY-1 population,but on AHSX-5 and AHTC-9 population,with 20.9%and 19.0%decrease of the GR₅₀ value respectively;similarly,the use of malathion(1000 g a.i.ha^-1)in combination with mesosulfuron-methyl had no major effect on HNXY-1population,but the GR₅₀ value of AHSX-5 and AHTC-9 population decreased 26.1%and 17.1%respectively.3.The gene fragment of plastid ACCase CT domain was amplified and sequenced,and all resistant shortawn foxtail populations possessed at least one ACCase target-site mutation.The following 12 populations has evolved the Ile-1781-Leu?ATA-TTA?mutation:AHSX-9,AHTC-1,AHTC-5,AHTC-6,AHDY-1,AHDY-3,AHHX-1,AHFX-1,JSJT-1,JSJR-1,JSJR-2,JSQT-1;The following nine populations has evolved the Ile-2041-Asn?ATT-AAT?mutation:AHSX-5,AHSX-6,AHSX-7,AHSX-8,AHSX-9,AHTC-2,AHTC-3,AHDY-3,JSHX-2;The following 19 populations has evolved the Asp-2078-Gly?GAT-GGT?mutation:AHSX-1,AHSX-2,AHSX-3,AHSX-4,AHTC-1,AHTC-9,AHDY-3,AHHX-2,AHLX-1,AHLX-2,JSHX-1,JSHX-3,JSHX-4,JSHX-5,JSHX-6,JSLS-2,JSJT-2,JSJT-3,JSGY-1;and the JSLS-1 population has evolved a very rare mutation Ile-2041-Thr?ATT-ACT?.The ALS gene fragment was also amplified and sequenced,and all resistant shortawn foxtail populations possessed at least one ALS target-site mutation.The following three populations has evolved the Pro-107-Arg?CCC-CGC?mutation:AHSX-5,AHSX-6,AHSX-7;The following five populations has evolved the Pro-107-Ser?CCC-TCC?mutation:AHSX-9,AHTC-7,AHTC-8,JSHX-2,SDTC-4;The following seven populations has evolved the Pro-107-Thr?CCC-ACC?mutation:AHSX-8,AHTC-2,JSHX-1,JSDH-1,JSDH-2,JSDH-4,JSQT-1;and the following 21 populations has evolved the Trp-574-Leu?TGG-TTG?mutation:AHSX-1,AHSX-2,AHSX-3,AHSX-4,AHSX-9,AHTC-1,AHTC-3,AHTC-5,AHTC-8,AHTC-9,AHDY-1,AHDY-3,AHHX-2,AHLX-1,AHLX-2,JSHX-3,JSHX-4,JSHX-5,JSHX-6,JSDH-3,JSGY-1.4.ALS activity was determined in vitro,compared with the wild type,the homozygous mutant ALS was insensitive to mesosulfuron-methyl.For the three mutations in Pro197 codon,their ALS enzymes had similar IC₅₀ values,which were 16.7²1.3 times of the wild type;the IC₅₀ value for ALS with the homozygous Trp-574-Leu mutation was much higher than others,and was 69.0 times of the wild type.5.Whole-plant dose-response experimrnts were conducted to determine the sensitivity to other herbicides using the purified populations.For shortawn foxtail,the ACCase Ile-1781-Leu mutation confered high resistance to fenoxaprop-P-ethyl and clodinafop-propargyl,moderate resistance to haloxyfop-P-methyl,clethodim,tralkoxydim and pinoxaden;the Ile-2041-Asn mutation confered high resistance to fenoxaprop-P-ethyl and clodinafop-propargyl,moderate resistance to haloxyfop-P-methyl,low resistance to pinoxaden,and no resistance to clethodim and tralkoxydim;the Ile-2041-Thr mutation confered high resistance to fenoxaprop-P-ethyl,moderate resistance to clodinafop-propargyl,low resistance to haloxyfop-P-methyl and pinoxaden,and no resistance to clethodim and sethoxydim;the Asp-2078-Gly mutation confered high resistance to fenoxaprop-P-ethyl and clodinafop-propargyl,moderate resistance to pinoxaden,low resistance to haloxyfop-P-methyl,clethodim and tralkoxydim.The ALS Pro-197-Arg and Pro-197-Thr mutation confered high,low and moderate resistance to mesosulfuron-methyl,pyroxsulam and flucarbazone-sodium respectively.The Pro-197-Ser mutation confered high resistance to mesosulfuron-methyl,moderate resistance to pyroxsulam and flucarbazone-sodium.The Trp-574-Leu mutation confered high resistance to mesosulfuron-methyl,pyroxsulamandflucarbazone-sodium.Isoproturon,33%diflufenican·flufenacet·flurtamone and F9600 could control the resistant shortawn foxtail effectively,with 100%control effect at 0.5×and 1.0×recommended rate in greenhouse.6.Three?d?CAPS assays were designed to detect mutations in ACCase 1781,2041 and2078 codon using EcoT22 I,EcoR I and EcoR V as the restriction enzyme respectively.A dCAPS assay was designed for specially detecting the Ile-2041-Thr mutation using BsoB I as the restriction enzyme.Two dCAPS assays were designed to detect mutations in ALS 197 and574 codon using BamH I and BstX I as the restriction enzyme respectively.The results of these?d?CAPS assays were reliable and were always in accordance with the sequencing results.7.The variance in seed germination and seedling emergence was compared between JSLS-1RR and JSLS-1SS populations under differernt light regime,temperature and buried depth.Results showed that light had no effect on seed germination of shortawn foxtail,the germination rate was always higher than 96%under different light regimes.Under constant temperature between 8²0°C and alternating 20/15°C,the maximum germinations of both JSLS-1RR and JSLS-1SS population were higher that 95%,and the former one had larger t_G50 values.Under constant 25°C,the maximum germination decreased,indicating that high temperature may inhibite shorawn foxtail germination.Under 0³.0 cm buried depth,most shortawn foxtail seedlings could emerge,and the emergence rate decreased gradually with the increase of buried depth.When the depth was 5.0 cm or more,both populations could not emerge.Under 1.0³.0cm buried depth,the maximum emergence of JSLS-1RR population was always lower than that of JSLS-1SS population.The resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl in shortawn foxtail from some regions of China was priliminarily investigated.Target-site mutation,which resulting in an insensitive target,was an important reason leading to resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl in shortawn foxtail.Moreover,the enhanced metabolism mediated by cytochrome P450 was account for the resistance in some populations,and different resistance mechanisms may coexist in a single shortawn foxtail plant.The resistant populations has evolved different cross resistance to other ACCase-and/or ALS-inhibiting herbicides,but is still sensitive to herbicides with other modes of action.The variance in seed germination and seedling emergence was compared under differernt light regime,temperature and buried depth by using the cosegregated subpopulations.Six?d?CAPS assays were developed to genotype ACCase and ALS at specified codon positions in shortawn foxtail.