Fenoxaprop-P-ethyl-resistance Alopecurus Japonicus In Wheat Fields And The Target-site-mediated Resistance

Alopecurus japonicus occurs in a majority of wheat and oilseed rape planting areas in China,and is one of serious weed species in winter crops in East China.Fenoxaprop-P-ethyl,a ACCase(acetyl-CoA carboxylase)-inhibiting herbicide,is one of the most important herbicides for controlling grass weeds including A.japonicus in wheat fields for many years.Recently,many growers complained the control efficacy of fenoxaprop-P-ethyl has decreased significantly.In order to improve the applying strategy of this herbicide,it is important to reveal the resistant situation of fenoxaprop-P-ethyl in A.japonicus and understand the relative resistance mechanisms.Target-site resistance(TSR)is one of the most commonly identified causes of ACCase-inhibiting herbicides resistance.Therefore,the main objectives of this study were to illuminate the sensitivity of A.japonicus poppulations in wheat fields in East China and reveal the TSR mechanisms of fenoxaprop-P-ethyl-resistant A.japonicus.Seed bioassay and whole-plant pot bioassay were conducted to evaluate the sensitivity to fenoxaprop-P-ethyl of 22 A.japonicus populations collected from Jiangsu and Anhui.Dose-response tests suggested that 16 populations were resistant to fenoxaprop-P-ethyl,while the other 6 populations were sensitive.According to ED50(effective dose of 50%inhibition in plant growth),the RI(resistance index)between the most resistant population(AHFD-1,ED50 = 1556.08 g a.i.·ha-1)and the most sensitive population(JNXW-1,ED50 =15.17 g a.i.·ha-1)was 102.59-fold.By contrast,the recommended field dose of fenoxaprop-P-ethyl used is 62.0 g a.i.ha-1.Whole-plant pot dose-response tests suggested that the population AHFD-1 was resistant to 5 other APP herbicides(clodinafop-propargyl,fluazifop-P-butyl,quizalofop-P-ethyl,haloxyfop-R-methyl and metamifop)and a PPZ herbicide(pinoxaden),but not to CHD herbicides(clethodim and sethoxydim).This population was not resistant to sulfosulfuron,mesosulfuron-methyl,flucarbazone-sodium,pyroxsulam,acetochlor,prometryne,chlorotoluron,trifluralin and glyphosate.Alopecurus japonicus was found to be a tetraploid by flow cytometer and chromosome counting,two commonly used methods in the determination of ploidy levels.We found that there are two copies of the gene encoding plastidic ACCase in A.japonicus and both the homologous genes are expressed.By comparing the sequences derived from the resistant populations and sensitive populations,the AHFD-1?AHFD-2 populations were found to contain a TGG-to-TGC mutation,which caused a tryptophan-to-cysteine mutation at codon position 2027,the AHFD-3 population was found to contain a GAT-to-GGT mutation,which caused a asparticacid-to-glycine mutation at codon position 2078,the JCJT-1?JCJT-2?JCJT-3populations were found to contain a ATA-to-CTA/TTA mutation,which caused a isoleucine-to-leucine mutation at codon position 1781,the JZJR-1 population was found to contain a ATT-to-AAT mutation,which caused a isoleucine-to-asparagine mutation at codon position 2041,the JLGY-4 poulation were found to contain a TGG-to-TGC mutation,which caused a tryptophan-to-cysteine mutation at codon position 1999,and a TGG-to-TTG mutation,which caused a tryptophan-to-leucine mutation at codon position 1999.This study found that the W1999C mutation conferred resistance to fenoxaprop-P-ethyl and moderate resistance to pinoxaden,but not to clodinafop-propargyl,haloxyfop-P-methyl,clethodim and sethoxydim.The W1999L mutation conferred resistance to fenoxaprop-P-ethyl,but not to clodinafop-propargyl,haloxyfop-P-methyl,clethodim and sethoxydim.These results testified the clear importance of the W1999C and W1999L mutations in conferring resistance to ACCase-inhibiting herbicides.ACCase of the W1999C-mutation plants was significantly less inhibited by fenoxaprop.The IC50(50%inhibiting concentration)for the wild-type plants was 0.46 ?M.However,the W1999C-mutation plants had a much higher IC50(4.58 ?M).The ACCase from the mutation plants was confirmed to be 9.96-fold insensitive to fenoxaprop.These results suggest that the resistance to fenoxaprop-P-ethyl in the mutation plants is due to the presence of an altered and insensitive ACCase.Differential gene expression analysis of a weed subjected to herbicide treatment is a key step towards more mechanistic studies.Such an analysis,often involving quantitative real-time PCR,requires suitable reference genes as internal controls.This study attempted to identity the reference genes in A.japonicus and compared the expression patterns of the ACCase gene,during herbicide treatment and under non-treatment conditions using these reference genes as controls.The stability of 8 commonly-used candidate reference genes(glyceraldehyde-3-phosphate dehydrogenase,GAPDH;ubiquitin,UBQ;capsine phosphatase,CAP;beta tubulin,TUB;eukaryotic initiation factor 4a,EIF4A;elongation factor-1 alpha,EF1;18S ribosomal RNA,18S;25S ribosomal RNA,25S)in roots,stems and leaves of fenoxaprop-P-ethyl resistant-or sensitive-plant individuals were tested using qPCR,in the situation with or without herbicide stress.The results were then further analyzed and ranked using geNorm,NormFinder and BestKeeper.EF1 and UBQ in roots;EF1,TUB,CAP and 18S in stems,and EF1,GAPDH and 18S in leaves were suitable references for the normalization.The expressions of these reference genes implied no significant difference in ACCase expressions in roots between W1999C-mutation(resistant)and wild-type(sensitive)plants.However,the ACCase expressions up-regulated in the stems and leaves of W1999C-mutation plants,compared to wild-type plants after fenoxaprop-P-ethyl application.The increased expression of ACCase may be involved in herbicide resistance in A.japonicus.Derived cleaved amplified polymorphic sequence(dCAPS)markers were developed to identify the target site of resistant mutations identified in this study.The technique of dCAPS was proven to be reliable and efficiency for detecting the resistance-endowing mutations in A.japonicus.The results showed that the frequencies of heterozygous W2027C mutation plants in AHFD-1 and AHFD-2 were 86.46%and 45.83%,respectively.The frequency of heterozygous D2078G mutation plants in AHFD-3 was 85.42%,the frequencies of heterozygous I1781L mutation plants in JCJT-1,JCJT-2 and JCJT-3 were95.83%,97.92%and 95.83%,respectively.The frequency of homozygous I2041N mutation plants in JZJR-1 was 91.30%,the frequencies of homozygous W1999C and W1999L mutation plants in JLGY-4 were 54.17%and 4.17%,respectively.This study characterize the sensitivity of 22 A.japonicus populations collected from Jiangsu and Anhui province to fenoxaprop-P-ethyl and the cross-resistance and multiple-resistance patterns in the most resistant population.A.japonicus was found to be a tetraploid,and there are two copies of the gene encoding plastidic ACCase and both the homologous genes are expressed.We also established the clear importance of the W1999C and W1999L mutations in conferring resistance to ACCase-inhibiting herbicides and the TSR mechanisms responsible for the resistance to fenoxaprop-P-ethyl in A.japonicus.The technique of dCAPS was developed and proven to be reliable and efficiency for detecting the resistance-endowing mutations in A.japonicus.