| Echinochloa crus-galli is one of the most severe weeds in rice field.Chemical control technique has long been applied to control E.crus-galli as the major method.E.crus-galli has become one of the worst resistant weeds in the world due to intensive application.It is a serious threat to agricultural production.Metamifop is a novel acetyl-coenzyme A carboxylase(ACCase)inhibitor that can effectivly control E.crus-galli,Leptochloa chinensis,etc.The aim of this research is to clarify dynamic variation of resistance to metamifop in E.crus-galli populations of Hubei Province and explore resistance mechanism of metamifop in E.crus-galli.In this study,resistance levels to metamifop in 49 E.crus-galli populations origined from Hubei Province and influence of resistance evolution by recurrent selection with different doses of metamifop in E.crus-galli were determinated.The populations(WX15,XY15,WX14-1,DY14-2,and ZS14-2)which exhibited high level resistance to metamifop and the resistant populations L3 and L4 generated by recurrent low-dose metamifop selection in initial population M0 were obtained.Mechanism of metamifop inhibition of ACCase CT domain in E.crus-galli was studied.Then,the field populations WX15 and XY15(RI=15.80,32.32)and low-dose herbicide selection populations L3 and L4(RI=42.50,49.92)were selected to study metamifop-resistant mechanism in E.crus-galli.This research is significant for scientific and rational use of metamifop and management in herbicide-resistant E.crus-galli.The results are as follows:1.Evaluation of resistance levels to metamifop in E.crus-galli in different regions of Hubei ProvinceWhole-plant assay tests were used to detect resistance levels to metamifop in 49 field populations of E.crus-galli collected from different regions of Hubei Province.The results demonstrated that the sensitivity of E.crus-galli to metamifop showed significant differences among these populations(Resistance index;RI=2.45-32.32).The populations(WX15,XY15,WX14-1,DY14-2,and ZS14-2)exhibited high level resistance to metamifop.The resistance levels gradually increased during 2011-2015.Combined with herbicide applications and toxicity correlation coefficient analysis,the toxicity of metamifop showed low correlation coefficient with penoxsulam(r=0.299,P=0.037)and bispyribac-sodium(r=0.293,P=0.041).There may be a certain resistance risk to metamifop in the E.crus-galli populations origined from the fields which were intensive application of ACCase inhibitors(cyhalofop-butyl,etc.)or ALS inhibitors(penoxsulam,bispyribac-sodium,etc.).2.Effect of different doses of metamifop on the evolution of herbicide resistance in E.crus-galliThe populations were obtained by recurrent selection with high-dose and low-dose metamifop in E.crus-galli initial population M0 for four rounds,respectively.Metamifop dose-response showed significant differences in the L2-L4 populations which generated by recurrent selection with low-dose metamifop compared with initial population M0.While metamifop dose-response in the populations selected with high-dose metamifop and untreated populations showed no significant differences with M0.These results indicated that recurrent selection with low-dose metamifop results in the rapid evolution of herbicide resistance in E.crus-galli.The resistance evolved in L2-L4 populations which was endowed by some factors.3.Interaction of metamifop with ACCase CT domain in E.crus-galliMetamifop is a noval ACCase-inhibition herbicide.Transmission electronic microscopy observation of metamifop-treated chloroplasts,detection of target enzyme activity in E.crus-galli,and molecular modeling,molecular docking,molecular dynamics simulations were used to study the influence of metamifop in target enzyme in order to explain the interaction of metamifop with ACCase CT domain in E.crus-galli.Metamifop treatment can severely destroy the ultrastucture of chloroplasts and inhibit the ACCase activity(IC50 = 41.1 n M)effectively in suscepitable E.crus-galli.Metamifop was bound in the active site of the CT domain of E.crus-galli ACCase at its dimer interface.The amino acids Tyr175,Phe391',Gly171,Thr194,and Lys201 played key roles in metamifop binding with CT domain in E.crus-galli.Tyr175 and Phe391' of the CT domain interacted with the oxazole ring in metamifop via ?-? interactions.The additional proton gained by protonation of metamifop was hydrogen-bonded to Gly171.When a large conformation altered in Tyr175 and Phe391',metamifop would gradually move from the binding pocket of the CT domain to the entrance of the binding channel,where it bound with Thr194 and Lys201.This research provides a foundation for elucidating molecular basis of target resistance and cross-resistance among ACCase herbicides,and for designing and optimizing ACCase inhibitors against weeds.4.Metamifop-resistant mechanism in E.crus-galliGenerally,single site mutation or multiple mutants of seven amino acid sites(Ile 1781,Trp 1999,Trp 2027,Ile 2041,Asp 2078,Cys 2088,Gly 2096)in the plant CT 2017 domain gene of chloroplastic ACCase confer ACCase inhibitor resistance.The CT domain genes from WX15,XY15,L3 and L4 populations include the seven sites without mutation.The results indicated that resistance to metamifop in these populations were likely endowed by non-target site resistance.Metabolic enzyme activities detections and metabolic enzymes reverse resistance tests demonstrated that non-target site resistance to metamifop in L3 and L4 populations were endowed by enhancing the expressions or activities of GSTs and P450 s.The contribution of GSTs was more than P450 s in herbicide resistance.The expression patterns of candidate resistance related genes were noted,and then proposed the metabolic pathway of metamifop in herbicide-resistant E.crus-galli.Gene expressions of P450 s,GSTs,GTs and ABC transporters showed significant differences in three stages of metamifop metabolic pathway in herbicide-resistant E.crus-galli compared with untreated susceptible E.crus-galli.Stage 1: Metamifop can be degraded into 6-CB and HPPFMA,then HPPFMA was further degraded into HPFMA and hydroquinone by Cytorome P450 s.The expressions of CYP2A6,CYP2C9,CYP71A1,CYP1A1,CYP1B1,CYP1A2,CYP71C1,CYP71C4,and CYP87A3 significantly increased in resistant biotypes compared with susceptible ones;down-regulated expression genes were CYP26 A,CYP71Z6,CYP97B2,and CYP93A2.Stage 2: 6-CB reacted with glutathione by GSTs.The production was hydrolysised to form a metabolite,then combinded with glucide by GTs.Hydroquinone and HPFMA combinded with glucide by GTs,respectively.Significant up-regulated expression genes were GSTF1,GST 23,GSTU6,UDP-glucuronosyltransferase 2B15,UDP-glucuronosyltransferase 1-1,UDP-glucuronosyltransferase 1-8;Down-regulated genes were GST 3 and GSTU20.Stage 3: The metabolites of stage 2 were transported to vacuole or apoplast by ABC transporters.Significant up-regulated expression genes were ABCC6 and ABCB1.These up-regulated metabolic genes were likely to play key roles in endowing metabolism-based herbicide resistance mechanism of metamifop in E.crus-galli. |
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