Study On The Resistance To Tribenuron-methyl In Water Chickweed (Myosoton Aquaticum L.moench.)in Wheat Fields

Water chickweed(Myosoton aquaticum(L.)Moench.)is a common and competitive broadleaf weed in wheat fields,which is widely distributed in most wheat planting regions,especially regions following a wheat-rice rotation in China.Tribenuron-methyl,a SU herbicide,was the most widely used herbicide to control broadleaf weeds in wheat fields in China due to its high efficacy.In recent years,farmers from many main wheat producing areas reported that water chickweed was more difficult to be controlled with tribenuron at recommended rate after this herbicide was continuously applied for many years.Water chickweed escapes have been becoming a threat to wheat production.The aims of the present study were: 1)to characterize the resistance level and identify the resistance mechanisms to tribenuron–methyl in many water chickweed populations collected from partial regions in China,2)to study the cross-resistance patterns of water chickweed to other ALS inhibitors and the sensitivity to alternative herbicides.As a result,to provide scientific guidline for the control of water chickweed.The sensitivity to tribenuron-methyl was tested in 43 water chickweed populations collected from Shandong,Henan,Anhui,Jiangsu,and Hubeiprovince.The mechanism for resistance in some populations was studied.The main results were as following:1.A single-dose screening test was used to identify the sensitivity to tribenuron-methyl in all the 43 populations.The application rate for treatment were 11.25,and 22.5 g ai ha-1,respectively.In total,25 populations were sensitive(S),while 18 populations were resistant(R)to tribenuron-methyl.The resistant lelve to tribenuron-methyl in 17 populations were characterised.Results showed that all the 17 populations have evolved high level of resistance to tribenuron-methyl.The resistance index(RI)were arranging from 94.7-fold to 546.7-fold when compared with S population.Especially,JS-17 population harveated from Suqian town,Jiangsu province showed the highest RI among all tested populations.2.Results from in vitro ALS assays indicated that ALS extracted from the plants of resistant populations was much less susceptible to tribenuron-methyl in comparison to the sensitivepopulation.Based on the the herbicide concentration resulting in 50% reduction of ALS activity(I50),The respective I50 value for R populations(SD-03,HN-02,HN-04,HB-01,AH-01,AH-02,AH-03,JS-07,JS-08,JS-16,and JS-17)were 28.9-fold to 75.5-fold greater than that of the S population.In addition,plants from HN-04,HB-01,and AH-01 populations express appropriately 1.8-2.4-fold total ALS activity tant that of S populations(JS-24,HN-03,SD-05).However,the total ALS activity was similar between the S and other R populations.3.The full length c DNA sequence of ALS gene from water chickweed is 2280 bp.It includes an open reading frame(ORF)of 2001 bp that encodes 666 amino acid residues,and the length of 5' untranslated sequence(UTR)is 60 bp,and 219 bp for 3' UTR.The accession numbers in Gen Bank were KF589890.1,and KF589891.1,respectively.4.To identify the molecular basis for resistance to tribenuron-methyl in water chickweed,a 1711 bp ALS gene fragment,including domain A to domain E,for S and R populations was amplified,sequenced,and and aligned.The analysis of nucleotide and deduced amino acid sequences showed that a subsititution located at position 197(corresponding to the Arabidopsis ALS amino acid sequence)ocurred in all the R populations.A Pro197 Glu mutation caused by nucleotide change of CCG to GAG was found in AH-02 population.The codon of position 197 changed from CCG to GCG/ACG which resulted in an Pro197Ala/Thr substitution in AH-03 population.A Pro197 Leu substitution resulted from CCG to CTG was indentified in SD-03,JS-07,and AH-01 population.Besides,the presence of two ALS gene mutations(Pro197Leu and Asp376Glu)was observed in AH01 individual,the substitution of Asp to 376 was caused by GAT to GAG mutation.The water chickweed populations HN-02,HN-04,HN-05,HN-06,HN-07,HB-01,HB-02,HB-03,JS-08,JS-16,and JS-17 possed a Pro197 Ser substitution which resulted from the codon change of CCG to TCG.5.A real-time quantification PCR assay was conducted to analyze the relative expression level of ALS gene of R and S populations by employing the ?-action gene as the keep-house gene.Results showed that there were no significant difference in the expression level of ALS gene between R and S populations.6.The GSTs activity toward CDNB for the untreated and treated plants from each population was analyzed over a period of 72 h.In untreated plants,there wo no significance on the GSTs avtivity for each population during 72 h.The GSTs extracted from the JS16 showed approximately 1.5-fold activity compared to the S during the experimental period.The plants from JS17 showed similar GSTs activity to that of JS24 over 72 h.However,the plants of HN-04,JS-07,and JS-08 populations express lower GSTs avtivity than S population.For treated plants,the GSTs activity could be induced by tribenuron-methyl for all tested populations.Especially,the induced GSTs activity of JS-16 and HN-04 is greater than S and other populations,and the GSTs activity of JS-16 is more rapidly induced by 48 h than S and other populations.7.The activity of P450 s in resistant populations was investigated by comparing the effective tribenuron-methyl rate causing a 50% growth reduction(GR50)in the absence and presence of P450 s inhibitor malathion.The results indicated that malathion significantly decrease the GR50 for tribenuron-methyl in JS-17 population.8.The cross-resistance experiments showed that R populations have evolved various resistance patterns to ALS inhibitors,however,sensitive to alternative herbicides.Results showed that SD-03,HN-02,HN-04,AH-01,AH-02,JS-08,JS-16,and JS-17 populations were highly resistant to pyrithiobac sodium,and the resistance index(RI)were from 11.0-to 109.2-fold,while HB-01,AH-03,and JS-07 were moderately resistant(5.0-to 9.2-fold)to pyrithiobac sodium.AH-02,JS-16,and JS-17 populations presented 26.9-,20.3-,and 16.6-fold resistance to florasulam,the RIs for populations SD-03,HN-02,HN-04,HB-01,AH-01,AH-03,JS-07,and JS-08 were arranging from 3.1-to 9.9-fold.The RIs for pyroxsulam in SD-03,HB-01,and AH-01 populations were 6.7-,4.5-,and 3.7-fold,when other tested R populations were highly resistant to pyroxsulam(1.1-fold to 52.9-fold).All the tested R populations have evolved high resistance to flucarbazone-Na(11.1-fold to 52.9-fold).AH-02 population showed 8.3-fold resistance to imazethapyr,however,all other R populations were still sensitive to imazethapyr.In addition,all tested populations were susceptible to 2,4-D butylate,fluroxypyr-meptyl,isoproturon,and diflufenican which possessing different modes of action.9.Molecular markers were designed in this study to rapidly identify the resistance as well as the specific mutations in ALS gene.The CAPS analysis were designed by utilizing Xho I,Dde I,Nru I,and Taq?I to detect the Pro197 Ser,Pro197Leu,Pro197 Ala,and Pro197 Glu mutation respectively.A restriction site of Apa I was made by introducing two mismatached bases in forward primer,then the d CAPS was used to detect the wild genetype.10.This research investigates the effect of various ALS resistance mutations in water chickweed on ALS functionality and plant growth.The enzyme kinetics of ALS from four purified water chickweed populations,each homozygous for the resistance mutations Pro197 Ser,Pro197Leu,Pro197 Ala,and Pro197 Glu,were characterized and the effect of these mutations on plant growth was assessed via relative growth rate analysis.All the resistance mutations populations express similar extractable ALS activity to wild populations,while the Pro197 Ser resistance mutation resulted in higher extractable ALS activity.In addition,all tested mutations have no significant effect on ALS kinetics.The Pro197 Leu mutation remarkably increased sensitivity to feedback inhibition by isoleucine and valine.The Pro197 Glu mutation slightly increased sensitivity to feedback inhibition by isoleucine.Moreover,all four mutations exhibited no significant effects on relative plant growth rate.However,all four purified water chickweed populations showed lower seed germination rate than wild populations(Pro197Pro > Pro197 Ser > Pro197Glu> Pro197 Leu >Pro197Ala).This study characterized the resistance to tribenuron-methyl in water chickweed populations collected from wheat fields of many main wheat producing areas.The target-site based resistance was established as being due to an insensitive form of ALS,resulting from substitutions at Pro197 in the ALS gene.The enhenced herbicide metabolism caused by the enhenced activity of GSTs and/or P450 s maybe associated with the tribenuron-methyl resistance in water chickweed.Cross-resistance experiments indicated that R populations exhibited various resistance patterns to other ALS inhibitors and alternative herbicides.Molecular markers were designed to rapidly identify the resistance as well as the specific Pro197 mutations individual in water chickweed population.In addition,this study priliminarily evaluate the effect of various ALS resistance mutations in water chickweed on ALS functionality and plant growth.