Study On The Resistance And Target-site Resistance Mechanism Of Chinese Sprangletop(Leptochloa Chinensis(L.) Nees) To Cyhalofop-butyl In Rice Paddy Fields

Chinese sprangletop(Leptochloa chinensis(L.)Nees)is a serious grass weed in rice paddies,just after Barnyard grass(Echinochloa crusgalliv).In recent years,with the popularization of light cultivation technology and the change of the environment in the world,Leptochloa chinensis has spreaded to the middle and lower reacher of the Yangtze River,which has seriously threatened the yield and quality of paddy field.Cyhalofop-butyl,an APP herbicide,has been primarily used as a post-emergence herbicide to successfully controlgramineous weeds,including L.chinensis in China.Hower,with the prolonged and widespread use of the same herbicide,the control efficacy of cyhalop-butyl to Leptochloa chinensis has decresed,even led to the development of resistance in this species.In order to clear whether or not Leptochloa chinensis has already produced resistance to cyhalop-butyl,identify the resistance level and explore the resistance mechanism of Leptochloa chinensis to cyhalop-butyl,this paper studies from four aspects,including the sensitivity of Leptochloa chinensisto cyhalop-butyl,the target-enzyme mechanism of resistance to cyhalop-butyl in Leptochloa chinensis,the cross-resistant and multi-resistant of thecyhalofop-butyl-resistant Leptochloa chinensis and the detection of a derived cleaved amplified polymorphic(dCAPS)assay in Leptochloa chinensis.The main results and conclusions are as follows:Whole-plant dose-response bioassay was used to determinethe susceptibility of 38 Leptochloa chinensis populations from different regions of Jiangsu,Zhejiang,Shanghai,Hubei and Zhejiang provinces to cyhalofop-butyl.The JHHA population,with a lower ED50 value of 1.89 g a.i.ha-1,was designated as the susceptible population.In contrast,the ZHYH population,with an ED50 value of 143.99 g a.i.ha-1,which was considerably higher than the recommended field dose of cyhalofop-butyl(90 g a.i.ha-1),was designated as the resistant population.The ratio suggested that ZHYH had 76.19-fold resistance to cyhalofop-butyl.In addition,the HHHM population from Hubei and the JHQP population from Jiangsu have also produced varying degrees of resistance to cyhalofop-butyl.The ED50 value were 64.82 and 55.81 g a.i.ha-1 and the RI wa up to 34.30 and 29.53.Finally,the JHHA population was selected as the sensitive population,and the ZHYH population was the resistant population.Two pairs of primerswere designed to amplify 873-and 531-bp DNA fragments encompassing the entire CT domain of L.chinensisACCase.By comparing the gene sequences of three resistant populations ZHYH?JHQP?HHHM and susceptible population JHHA,the results showed that there were mutations of ACCase amino acids in the populations of three species:The 2027th amino acids of ACCase mutated from tryptophan(Trp)to cysteine(Cys)in ZHYH and JHQP populations.This mutation changed TGG to TGC only in the resistant population.Trp-1999-Ser and Trp-1999-Leu substitutions were found in HHHM population.W1999S was changed by TGG to TCG and W1999L was changed by TGG to TTG.This is the first report on the molecular basis underlying cyhalofop-butyl resistance in L.chinensis.This mutation may be the main cause of the resistance to cyhalofop-butyl.In assays of ACCase activity in Leptochloa chinensis revealed the ACCase activity of the W2027C resistant Leptochloa chinensispopulation ZHYH and sensitive Leptochloa chinensis population JHHA decreased in different degree with the increase of the concentration of Cyhalofop acid,but the decrease of ACCase activity in sensitive Leptochloa chinensis was more obvious.Besides,in ZHYH,the sensiticity of ACCase activity to cyhalofop-butyl was lower,the IC50 of ZHYH was 16.91 ± 1.49 ?M,which was 5.25-flod greater than the IC50 of JHHA.The results indicated that the difference of ACCase activity between the resistant and susceptible populations to cyhalofop-butyl may be one of the reasons for the resistance of Leptochloa chinensis to cyhalofop-butyl.The expression levels of ACCase gene between ZHYH and JHHA were measured by qPCR method.After the cyhalofop-butyl treatment,the expression of ACCase between cyhalofop-butyl-resistant Leptochloa chinensisand susceptible Leptochloa chinensis have no significant difference.The resistant ZHYH population and the resistant HHHM population produced different resistance patterns to other herbicides,whereas the susceptible JHHA population was controlled effectively by all tested herbicides.The ZHYH population with W2027C mutation expressed resistance to APP herbicides such as metamifop,clodinafop-propargyl,fenoxaprop-P-ethyl,quizalofop-P-ethyl,and fluzifop-P-butyl as well as DEN herbicides such as pinoxaden,but not to CHD herbicides such as sethoxydim and clethodim.The HHHM population withtwo mutants of W1999s and W1999L poduced cross-resistance to fenoxaprop-P-ethyl which has the same mechanism with cyhalofop-butyl.At the same time for the other APP herbicides Metamifop,Clodinafop propargyl,quizalofop-p-ethyl,fluazifop-p-butyl and DEN herbicide pinoxaden showed sensitivity decreased.But this population also has no resistance to CHD herbicides.There were no multiple resistances in both ZHYH and HHHM populations to the acetolactate synthase-inhibiting herbicides pyribenzoxim and bispyribac-sodium,the protoporphyrinogen oxidase herbicide oxyfluorfen,and the urea herbicide isoproturon.However,the resistant population was probably only slightly resistant to hormonal herbicides quinclorac.In addition,a dCAPS assay was developed to rapidly detect the W2027C mutation.The W2027C primer introduced two mismatches to create a restriction site for HhaI.The primer amplified a 265-bp DNA fragment.This method is very useful for detecting sequence polymorphisms.After restriction digestion with HhaI,the mutant sequences were cut into a 201-bp and an undigested 64-bp fragment,but WT sequences were not cut.The mutant sequences were classified as homozygous or heterozygous.The heterozygous ACCase loci produced both the 201-and 265-bp fragments,whereas the homozygous mutant ACCase loci produced a single 201-bp fragment.The successful development of the dCAPS assay was confirmed by randomly analyzing 100 plants from the resistant ZHYH population.The results indicated 52 RS and 48 SS plants,but no RR plants.In this paper,the levels of resistance of L.chinensis to cyhalofop-butyl were determined.This is the first report on the molecular basis underlying cyhalofop-butyl resistance in L.chinensis.We found the W2027C,W1999S and W1999L three mutations in the ACCase gene of cyhalofop-butyl-resistant L.chinensis.We also investigated the cross-and multi-resistance of the cyhalofop-butyl-resistant L.chinensis carrying different mutations.Simultaneously,we developed a dCAPS method for detecting the W2027C mutation and the frequency of its occurrence in the ACCase gene of L.chinensis rapidly and accurately.