Herbicide Resistance In Alopecurus Aequalis From Wheat Fields In Anhui Province And Rapid Detection Of Its Resistance Mutations

Wheat(Triticum aestivum L.)is one of the most important cereal crops worldwide,ranking third in the planting area among the three major grain crops in China.Its production has always been threatened by various harmful organisms in the fields.Shortawn foxtail(Alopecurus aequalis Sobol.)is a common and malignant arable weed,widely distributed in rice-wheat rotation fields and rapeseed fields in the middle and lower reaches of the Yangtze River in China.Its strong tillering and competitive ability can cause significant yield reduction or even complete crop failure.In recent decades,weed control in wheat fields has mainly relied on post-emergence herbicides fenoxaprop-P-ethyl and mesosulfuron-methyl,which act on the acetyl-Co A carboxylase(ACCase)and acetolactate synthase(ALS)of weeds,respectively.They are broad-spectrum,high efficiency,low toxicity,and are relatively safe.However,their single mode of action(MOA)is prone to induce weed resistance through long-term repeated use.Anhui Province is one of the major wheat-producing provinces in the middle and lower reaches of the Yangtze River in China.Currently,farmers in this region generally report that commonly used herbicides at recommended field doses are no longer effective against A.aequalis in wheat fields,indicating the possible development of herbicide resistance in this weed.Therefore,this study investigated the occurrence of A.aequalis and collected suspected herbicide-resistant populations in the main wheat-growing areas of Anhui Province.Based on this,resistance identification and mechanism research were conducted on different populations,and the herbicide resistance pattern of different resistant populations was determined.In addition,a rapid detection method for common resistance mutations in the target gene ACCase of A.aequalis was established using molecular marker technology,aiming to provide scientific basis for monitoring and controlling herbicide resistance in A.aequalis in wheat fields in Anhui Province.The main results of this study are as follows:1.Population collection,and resistance identification of A.aequalis to commonly used herbicidesThe occurrence of A.aequalis was investigated and suspected resistant populations were collected in the main wheat planting areas of Anhui Province.A total of 18 populations were collected,which were mainly distributed in Tianchang,Lai’an,Fengyang,Sixian,Shouxian,Yingshang,Fengtai,and other regions.Based on each population,the herbicide susceptibility was tested by using the field recommended doses of fenoxaprop-P-ethyl(62 g a.i.ha-1)and mesosulfuron-methyl(9 g a.i.ha-1),respectively.The results showed that 13 populations had developed herbicide resistance,among which AHMG-1 population only developed resistance to mesosulfuron-methyl,while AHLA-2,AHLA-4,AHTC-2,AHTC-3,AHFY-2,HNSX-1,SZSX-2,AHHQ-4,AHFT-1-2,AHFT-2,AHFT-3 and AHFT-4 populations developed multiple resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl.For all resistant populations,the herbicide resistance levels of different populations were determined by using greenhouse pot method at the whole-plant level.The results showed that 12 multiple resistant populations all developed high level resistance to fenoxaprop-P-ethyl,with resistance indexes(RIs)ranging from 18.92 to 844.53,among which AHTC-3 population had the highest RI of 844.53;13 mesosulfuron-methyl resistant populations also developed high level resistance,with RIs ranging from 19.75 to 96.02.2.Study on the target-site based resistance mechanism of A.aequalis to fenoxaprop-P-ethyl and mesosulfuron-methylFor the 12 fenoxaprop-P-ethyl-resistant populations,the target gene ACCase was amplified and sequenced for comparative analysis.The resistance mechanism of A.aequalis to fenoxaprop-P-ethyl was studied in the 12 fenoxaprop-P-ethyl-resistant populations by amplifying and sequencing their target gene ACCase.The results showed that all 12 populations had ACCase mutations.Three populations,including AHTC-2,AHFY-2,and AHFT-2,had a mutation of Ile-1781-Leu;four populations,including AHLA-4,AHTC-3,HNSX-1,and AHFT-1-2,had a mutation of Ile-2041-Asn;two populations,including SZSX-2 and AHFT-3,had a mutation of Asp-2078-Gly;and three populations,including AHLA-2,AHHQ-4,and AHFT-4,had two different forms of mutations in different plants,namely Ile-2041-Asn or Asp-2078-Gly.For the mesosulfuron-methyl-resistant populations,the full-length sequences of the two copies of the target gene ALS,ALS1 and ALS2,were amplified and sequenced for comparative analysis.The results showed that except for the AHFT-3 population,all other12 resistant populations had ALS mutations.Five populations had mutations only in ALS1,namely AHTC-3,HNSX-1,and AHFT-1-2 at Pro-197-Thr in ALS1,and AHMG-1 and AHHQ-4 at Trp-574-Leu in ALS1.In addition,three populations had mutations only in ALS2,namely AHTC-2 at Pro-197-Thr,AHFY-2 at Pro-197-Ser,and SZSX-2 at Trp-574-Leu in ALS2.Three populations had mutations in both ALS1 and ALS2 at position197 or 574.The remaining AHLA-2 population had two different mutations in ALS1 in different plants,namely Pro-197-Thr or Trp-574-Leu.3.Study on the susceptibility of A.aequalis populations carrying different ACCase resistance mutations to other herbicidesThe herbicide resistance patterns of three fenoxaprop-P-ethyl-resistant populations with different ACCase mutations,AHFT-2(I1781L),AHLA-4(I2041N),and AHFT-3(D2078G),were determined for other herbicides.The results showed that all populations had varying levels of cross-resistance to ACCase inhibitors clodinafop-propargyl,sethoxydim and pinoxaden,but were more susceptible to clethodim.All populations showed high-level multiple resistance to four different categories of ALS inhibitors,imazethapyr,pyroxsulam,pyrithiobac-sodium and flucarbazone-sodium,with RIs up to383.33 times,and the high-level ALS resistance in AHFT-2 and AHLA-4 populations may be related to their ALS mutations.In addition,all populations showed multiple resistance to the Photosystem II(PSII)inhibitor isoproturon,and AHFT-2 and AHFT-3 populations also showed multiple resistance to the 1-deoxy-d-xylulose 5-phosphate synthase(DXS)inhibitor bixlozone,while the AHLA-4 population remained susceptible to bixlozone.4.Establishment of a rapid detection method for ACCase mutations in A.aequalis resistant to fenoxaprop-P-ethylMolecular markers were successfully established using(derived)cleaved amplified polymorphic sequence(d CAPS)technology for each resistance mutation of three A.aequalis populations carrying different ACCase resistance mutations,AHTC-2(I1781L),AHLA-4(I2041N),and SZSX-2(D2078G).Markers targeting the resistance mutation sequence were established for AHTC-2 and SZSX-2 populations,while a marker targeting the wild-type sequence was established for the AHLA-4 population.Analysis showed that each individual ACCase of the AHTC-2 population had heterozygous mutations,while each individual ACCase of the AHLA-4 population had homozygous mutations,and the SZSX-2 population had individuals with homozygous or heterozygous ACCase mutations.Furthermore,a rapid detection method for the AHLA-4(I2041N)resistance mutation was successfully established using loop-mediated isothermal amplification(LAMP)technology,which allows direct observation of color change by the naked eye to determine whether the target ACCase gene mutation has occurred in A.aequalis plants.Compared to the traditional polymerase chain reaction(PCR),the LAMP-based detection method has a sensitivity that is approximately 1000 times higher.In summary,this study clarified the population of A.aequalis in major wheat producing areas of Anhui Province was collected through field investigations and determined its susceptibility and resistance levels to the commonly used herbicides fenoxaprop-P-ethyl and mesosulfuron-methyl through single-dose screening and resistance level determination.The resistance mechanism of resistant populations was preliminarily elucidated by target gene sequencing,and herbicide resistance patterns were determined for populations carrying specific ACCase resistance mutations.Finally,molecular detection techniques for specific ACCase mutations were successfully established using(d)CAPS and LAMP technologies.The results obtained from this study are expected to provide a scientific basis for monitoring and management of ryegrass resistance in wheat fields in Anhui Province.