Study On The Resistance Mechanism Of Echinochloa Crus-galli(L.)P.Beauv.to Penoxsulam And Cyhalofop-butyl

Echinochloa spp.,are common and malignant weeds in paddy fields,among which Echinochloa crus-galli（L.）P.Beauv.has a widespread distribution and serious threat.Chemical weed control is the main measure for controlling weeds in paddy fields.Penoxsulam and cyhalofop-butyl are the most commonly used herbicides in paddy fields,both of which function as inhibitors of acetolactate synthase（ALS）and acetyl-coenzyme A carboxylase（ACCase）,respectively.However,the limited action sites make them susceptible to decreased control efficacy and resistance risks after long-term continuous usage.In order to clarify the resistance mechanisms of Echinochloa spp.to penoxsulam and cyhalofop-butyl,this study examined the resistance levels and mechanisms of 45 populations of Echinochloa spp.from various regions of China.The main results are as follows:1.The results of the single-dose assay showed that among the 39 populations,there were 27populations resistant to penoxsulam,33 populations resistant to cyhalofop-butyl,and 22 populations resistant to both penoxsulam and cyhalofop-butyl.The resistance level assay results showed that 12 E.crus-gulli populations had evolved high-level resistance to penoxsulam,with resistance indexs ranging from 11.43 to 94.86,accounting for 50.0%of the 24 tested populations.12 E.crus-gulli populations had evolved high-level resistance to cyhalofop-butyl,with resistance indexs ranging from 11.11 to 78.60,accounting for 60.0%of the 20 tested populations.2.The results of target enzyme ALS gene sequencing showed that E.crus-galli.had 3 ALS allele genes,and 3 different ALS gene mutations were found in the tested populations,namely Trp-574-Leu,Pro-197-Leu,and Pro-197-His.The Pro-197-His mutation has not been reported in E.crus-galli.The ALS gene expression levels of the resistant populations AH04,HLJ02,JL01 with different ALS gene mutations,the resistant population JS01 without mutation,and the sensitive population YN01 were measured before and after treatment with penoxsulam.The results showed no significant difference in ALS gene expression levels between the resistant populations AH04,HLJ02,JL01,JS01,and the sensitive population YN01before and after treatment with penoxsulam.3.The results of in vitro ALS activity assay showed that before treatment with penoxsulam,there was no significant difference in total ALS catalytic activity between the resistant populations AH04,HLJ02,JL01,JS01 and the sensitive population YN01.However,after treatment with penoxsulam,the I₅₀ value for the resistant populations AH04,HLJ02,JL01,and JS01 was 4.12,1.11,1.04,and 1.38 times higher than that for the sensitive population YN01.The resistance of the AH04 population to penoxsulam was related to the decreased sensitivity of its target enzyme ALS to penoxsulam.4.Homology modeling and molecular docking results showed that the Trp-574-Leu mutation would cause the loss of theπ-πinteraction between penoxsulam and Trp,resulting in a decrease in the binding affinity between penoxsulam and ALS.The Pro-197-Leu and Pro-197-His mutations resulted in changes in the interaction and active channel shape between penoxsulam and ALS,leading to decreased binding affinity.Using insect cell heterologous expression of WT＿ALS643,WT＿ALS644,Trp-574-Leu,Pro-197-Leu,and Pro-197-His ALS proteins,and treating them with 1μmol·L^-1 penoxsulam,the activity inhibition rates for the three mutant ALS proteins were 35.40%,48.25%,and 42.86%,significantly lower than that for the wild type.The molecular docking and protein level results demonstrated that the ALS gene mutations,including Trp-574-Leu,Pro-197-Leu,and Pro-197-His,result in resistance to penoxsulam.5.The sequencing results of the target enzyme ACCase gene showed that E.crus-gulli has 6 ACCase allele genes,and no ACCase gene mutations were found in the tested populations.ACCase gene expression levels were measured in the resistant population JS01 and the sensitive population YN01,and the results showed no significant difference in ACCase gene expression levels before and after treatment with cyhalofop-butyl.6.The activity analysis results of P450s and GSTs showed that the P450s inhibitor malathion had no significant effect on the sensitivity of JS01 population to penoxsulam and cyhalofop-butyl.Compared with the sensitive population YN01,the GSTs activity of the resistant population JS01 did not show a significant difference after treatment with penoxsulam,but the GSTs activity of JS01 was significantly increased at 3 d and 5 d after treatment with cyhalofop-butyl.7.Metabolic difference analysis results showed that compared with the sensitive population YN01,the resistant population JS01 did not show significant differences in absorption of penoxsulam and cyhalofop-butyl,but the metabolic rates of the two herbicides were significantly accelerated in the JS01population.The JS01 population exhibited metabolic resistance to penoxsulam and cyhalofop-butyl.8.The results of fluorescence quantitative PCR combined with transcriptomics analysis showed that18 differentially expressed genes associated with metabolic resistance to penoxsulam were identified in the resistant population JS01,and 11 differentially expressed genes associated with metabolic resistance to cyhalofop-butyl were also identified.Both the CYP86B1-like and glutathione S-transferase 2 genes may be involved in the metabolic resistance of JS01 population to penoxsulam and cyhalofop-butyl.In summary,this study clarified the resistance levels and degree of multiple resistance of E.crus-gulli to penoxsulam and cyhalofop-butyl in various regions of China;identified the differences in target genes,and for the first time,discovered the Pro-197-His mutation in E.crus-gulli.The study also demonstrated that ALS gene mutations were one of the reasons for the resistance of E.crus-gulli to penoxsulam,verified at the molecular docking and protein levels.The study also identified the absorption and metabolic differences of JS01 population to penoxsulam,cyhalofop-butyl,and metamifop.By using q RT-PCR combined with transcriptomics analysis,a batch of differentially expressed genes that are involved in the metabolic resistance of JS01 population to penoxsulam and cyhalofop-butyl were preliminarily screened.These research results reveal the resistance levels and molecular mechanisms of E.crus-gulli to penoxsulam and cyhalofop-butyl,and provide a reference for the diagnosis and integrated management of resistant weeds,as well as the development of new herbicides.