| Flixweed(Descurainia sophia L.),an annual or biennial cruciferae herb,is one of the malignmant weeds in wheat field in China.Tribenuron-methyl,a sulfonylureas herbicide,which was registered in 1988 in China,has been used to control broadleaf weeds.The intensive and persistent application has made flixweed develop high resistance to tribenuron-methyl.In this study,the tribenuron-methyl resistance level and resistance mechanisms of flixweed populations collected from Beijng,Henan,Hebei and Shandong provinces were investigated,and the subpopulations with different ALS mutations were obtained by reproduction of individual plants.The effects of different ALS mutations on cross-resistance patterns,ALS functionality and branched amino acid concentrations were researched,and a rapid detection method of resistant flixweed by molecular biology was discussed.The main results were summarized as follows:1.The determination of tribenuron-methyl-resistant flixweed populationsThe tribenuron-methyl resistance of 73 flixweed populations was determinated by discriminating dose assay,the results indicated that 28 populations have evolved resistance to tribenuron-methyl,18 populations have high risk of developing resistance to tribenuron-methyl,and 27 populations were susceptible to tribenuron-methyl.2.The ALS mutation diversity in resistant flixweed populationsThe results of ALS mutation diversity revealed two ALS genes,named as ALS1 and ALS2,existed in flixweed.Amino acid substitutions at proline(197)into leucine,serine,threonine,histidine and tyrosine,tryptophan(574)into leucine or aspartic acid(376)into glutamate in ALS1 gene were identified,and the proline(197)changed to serine or threonine was also found in ALS2.The percentages of Pro-197-Leu,Pro-197-Ser,Pro-197-Thr,Pro-197-His,Pro-197-Tyr,Asp-376-Glu and Trp-574-Leu mutation were 39.3%,28.6%,28.6%,10.7%,3.6%,3.6%and 7.15 respectively in total 28 resistant mutation populations.In addition,Pro-197-Tyr mutation was firstly identified and found in flixweed,and Trp-574-Leu mutation(in ALS1)and Pro-197-Thr mutation(in ALS2)were observed in a single plant,which was founded firstly in field-evolved resistant weeds.3.The effects of different ALS mutations on resistance and cross-resistanceIn order to reduce the difference of genetic background,the purified pHB08,pHB22,pHB23,pHB24,pHB25,pHB42 and pHB30 subpopulations with different ALS mutations were obtained by reproduction of individual plants,the pHB08,pHB22,pHB23,pHB24,pHB25 and pHB42 subpopulations were homozygous for Pro-197-Leu,Pro-197-Ser,Pro-197-Thr,Pro-197-His,Asp-376-Glu and Trp-574-Leu in ALS1 respectively,the pHB30 subpopulation for Trp-574-Leu in ALS1 and Pro-197-Thr in ALS2.The whole-plant response experiments established the purified pHB08,pHB22,pHB23,pHB24,pHB25,pHB42 and pHB30 subpopulations displayed high resistance to tribenuron-methyl,and the pHB30(Trp-574-Leu + Pro-197-Thr)and pHB25(Asp-376-Glu)subpopulations exhibited the higher resistance levels compared with pHB08,pHB22,pHB23,pHB24 and pHB42 subpopulations(789.3-815.0 vs 152.3-366.3).The results of ALS-inhibiting herbicide cross-resistance endowed by specific ALS mutations indicated the subpopulations carrying a Pro-197-Leu?Pro-197-Ser?Pro-197-Thr or Pro-197-His mutation exhibited cross-resistance to halosulfuron-methyl(SU),flumetsulam(TP),flucarbazone-Na(SCT),but not to imazethapyr(IMI)and pyribenzoxim(PTB),while the pHB25 with Asp-376-Glu mutation,the pHB42 with Trp-574-Leu mutation and the pHB30 with two mutated ALS gene displayed cross-resistance to all five ALS-inhibiting herbicides.4.The effect of different ALS mutations on ALS functionalityThe results of ALS activity and sensitivity to tribenuron-methyl determination showed the sensitivity to tribenuron-methyl of ALS extracted from pHB08,pHB22,pHB23,pHB24,pHB25 and pHB42 subpopulations decrease 250.0,205.8,263.7,708.4,2844.7 and 485.3 folds compared with that of susceptible populations,and ALS from resistant populations with Pro-197-Leu,Pro-197-Ser,Pro-197-Thr or Pro-197-His mutation was found to display lower ALS activity(11%-21%)than the S population,the population with Asp-376-Glu mutation displayed higher ALS activity(26%)than the S,and the population with Trp-574-Leu mutation had a similar ALS activity with the S.The results of enzyme kinetics determination indicated that ALS Km and Vmax values for resistant subpopulations pHB08,pHB22,pHB23,pHB24,pHB25 and pHB42 were generally lower than for that of S population,Km values decreased by 0.63-0.87 times,and Vmax values decreased by 0.30-0.91 times.Studies on feedback inhibition established populations with Pro-197-Leu,Pro-197-Ser,Pro-197-Thr or Pro-197-His mutation caused decrease in ALS sensitivity to leucine,and unchanged ALS sensitivity to valine and isoleucine,the ALS of Asp-376-Glu or Trp-574-Leu mutation populations were less sensitive to feedback inhibition compared to that of S population by leucine,valine and isoleucine.5.The effect of different ALS mutations on branched chain amino acid concentration in flixweedThe results of branched-chain amino acid concentrations determination in flixweed with different ALS mutations indicated that there was no difference in leucine and isoleucine concentrations from resistant plants with Pro-197-Leu,Pro-197-His,Pro-197-Ser or Pro-197-Thr mutations and susceptible plants(isoieucine concentration is slightly lower in plants with Pro-19/-Ser mutation than S plant).Leucine and valine concentrations were slightly higher in Asp-376-Glu or Trp-574-Leu mutation plants than S plants.6.A rapid dectection method of tribenuron-methyl-resistant flixweedBased on the base difference at Pro 197,Asp376 or Trp574 between susceptible and resistant plants,BsmFI,Mbol and BsrDI restriction digestion of CAPS were developed to identify the target-site resistance of 48 flixweed plants.The results indicated the accuracy rate of this method was 100%,and could be used to determinate the resistant flixweed plants with ALS mutations. |
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