Study On The Resistance Mechanism Of Ipomoea Nil To Fomesafen

Soybean is an important oilseed and food crop in China.Ipomoea nil（L.）Roth is a common broadleaf weed in soybean fields in Liaoning Province,China.It possesses competitive biological attributes,which hinder mechanized harvesting and limit the yield and quality of soybean.Fomesafen is a protoporphyrinogen oxidase（PPO）inhibitor herbicide widely used to control broadleaf weeds in soybean fields such as I.nil.However,the control efficacy of fomesafen has decreased under successive and long-term application in many soybean planting areas.Based on this,this study collected 66 populations from 8 areas in Liaoning Province with severe damage caused by I.nil including Dalian,Jinzhou,Huludao,Chaoyang,Fuxin,Shenyang,Yingkou,and Dandong.This study clarified the resistance levels of I.nil to fomesafen and explored the resistance mechanism.Meanwhile,the seed germination adaptability of resistant I.nil was also investigated.These researches were conducted for the purpose of providing a scientific basis and theoretical guidance for the rational prevention and control of I.nil,as well as for delaying the spread and evolution of resistance.The results were listed as followed:1.34 I.nil populations have developed varying degrees of resistance to fomesafen,among which the DL04 population was the heaviest one and has developed cross and multiple resistance.The discriminate dosage method was conducted to preliminary screen the resistance of all collected populations.The result showed that 32 sensitive populations such as SY01,SY02,and HL05,as well as 34 resistant populations such as DL01,DL02,and DL04 were detected,respectively indicating that most areas of Liaoning have shown resistance to fomesafen.To further clarify the resistance levels,the whole-plant bioassay method was used to evaluate the sensitivity of all resistant populations and two sensitive populations（SY01 and SY02）to fomesafen.The results showed that all resistant populations exhibited varying degrees of resistance to fomesafen,with a resistance index ranging from 2.79 to 21.03.among them,47.06%were recognized as high-level resistance,medium resistance populations,and low resistance each contributed 26.47%of the total.DL04 population with the highest resistance level was selected as the research object for measuring the sensitivity to other herbicides by whole plant bioassay.The results showed that compared to the susceptible population SY01,the resistant population DL04 exhibited moderate levels of resistance to PPO inhibitor herbicides lactofen and flumioxazin（5.73-8.03）;High levels of resistance to the ALS inhibitor thifensulfuron-methyl（19.80）;Fortunately,DL04 is still sensitive to the synthetic hormone herbicide 2,4-dioctyl ester and the PS II inhibitor bentazone.2.Gene mutation and reduced sensitivity of target enzyme were not involved in the fomesafen resistance.This study amplified and sequenced the encoding regions of target genes PPX1 and PPX2 in the I.nil populations and did not detect any reported target mutation forms that could lead to resistance to PPO inhibitor herbicides.The result of in vitro PPO activity assay also showed that no significant difference between resistant and susceptible populations.ACT and GAPDH were selected as stable expression internal reference genes after comprehensive analysis by ge Norm,Best Keeper,Norm Finder,and Re Finder.Further research was conducted on the expression levels of PPX1 and PPX2 in resistant and susceptible populations.The results showed that the application of fomesafen can induce the expression of PPX1 and PPX2 genes in DL04,which was 4.19-fold and 9.29-fold compared with SY01respectively.3.Enhanced metabolic detoxification and antioxidant stress ability was the important reason for the non-target-site resistance of the DL04 population to fomesafen.This study investigated the differences in the activity of P450s and GSTs in susceptible and resistant populations through metabolic enzyme inhibitors.The results showed that pretreatment with GSTs inhibitors 4-chloro-7-nitro-2,1,3-benzoxadiazole（NBD-Cl）had no significant effect on the sensitivity of the resistant population DL04 to fomesafen,while pretreatment with P450s inhibitors malathion and piperonyl butyl ether（PBO）reduced the GR₅₀ value of DL04 by 50.85%and 25.39%,respectively.HPLC-MS/MS was used to determine the absorption and metabolism levels of the resistant and susceptible I.nil populations.The results showed that the metabolic rate of the resistant population DL04 was much higher than that of the sensitive population SY01.However,after synergistic application of malathion,the metabolic rate of the resistant population was significantly suppressed.The above results confirmed that the resistant population DL04 has evolved metabolic resistance mediated by P450s.In addition,compared with SY01,the content of ROS and MDA in the resistant population DL04 was lower,while the activities of antioxidant enzymes SOD and POD were significantly higher after treatment with fomesafen.This indicates that the enhancement of antioxidant stress ability is also one of the non-target-site resistance mechanisms of DL04.4.P450s,GTs,ABC transporters,and peroxidase genes related to detoxification metabolism and antioxidant stress capacity are responsible for the metabolic resistance of DL04to fomesafen.In this study,the resistant population DL04 and the susceptible population SY01were taken as the research objects for transcriptome sequencing to screen candidate genes related to the fomesafen metabolic resistance.The results showed that 36887 differentially expressed genes were generated in four comparison groups（RCK＿SCK;RT＿ST;RCK＿RT;SCK＿ST）,focusing on the gene family related to herbicide metabolism and detoxification,and31 genes possibly related to the metabolic resistance of fomesafen were screened by combining the expression and significance of the differentially expressed genes.Sequencing samples and parallel samples were used for two rounds of q RT-PCR validation.It was determined that a total of four P450s,two GTs,three ABC transporters,and two peroxidase genes may be related to the metabolic resistance of DL04 to fomesafen.5.Different tolerance abilities to p H and water stress in the seed germination were observed in resistant and susceptible I.nil populations.This study investigated the adaptive differences between the resistant population DL04 and the susceptible population SY01 under different temperatures,photoperiod,p H,water stress,salt stress,and burial depth conditions.The results showed that the sensitive population SY01 had a high germination rate at p H 4 to10 and was sensitive to water stress;However,the germination rate of the resistant population DL04 significantly decreased when the p H was greater than 8,and its sensitivity to water stress was lower.In addition,there were no significant differences in the germination of resistant and susceptible I.nil in terms of photoperiod,temperature,salt stress,and burial depth.They were not sensitive to photoperiod and could germinate under a wide range of temperature conditions with strong salt tolerance.Moreover,their emergence was severely inhibited when the burial depth was greater than 3.2cm.