Resistance Mechanism Of Lithospermum Arvense L. To Tribenuron-methyl In Wheat Fields

Lithospermum arvense L.,an annual or biennial Boraginaceae weed,is a fast-spreading and malignant broadleaf weed widely distributing in wheat fields in Huang-Huai-Hai region.Tribenuron-methyl,a sulfonylureas herbicide developed by DuPont Company of America,was introduced in 1988 to selectively control broadleaf weeds in wheat fields in China.Unfortunately,in recent years,L.arvense was more difficult to be controlled with tribenuron at recommended rate after this herbicide was continuously applied for several years.L.arvense has caused serious damage in yield and quality of wheat.The aims of the present study were to characterize the sensitivity of 22 L.arvense populations collected from Henan,Shandong,Anhui and Shaanxi Province in China to tribenuron–methyl.Meanwhile,the resistance mechanisms to tribenuron–methyl and cross resistance pattern to other acetolactate synthase（ALS）inhibitors and alternative herbicides in some L.arvense populations was identified.As a result,to provide scientific guideline for the control of L.arvense.The main results were as follows:1.A single-dose screening test was used to identify the sensitivity to tribenuron-methyl in all the 22 populations.The application rate for treatment was 18 g a.i.ha^-1 and 13 resistant（R）populations and 9 susceptive（S）populations were identified.Further whole-plant dose–response experiments was conducted to determined the resistance levels of R populations above.The results showed that the resistance levels to tribenuron-methyl were significantly different in all R populations.The resistance index（RI）were arranging from4.5-fold to 349.6-fold when compared with S population.Especially,HNXX-1 population collected from Xinxiang city,Henan province showed the highest RI among all tested populations.2.The L.arvense ALS gene fragments of 13 R populations and 3 S populations were amplified and sequenced and only HNXX-1 population possessed ALS target-site mutation of Trp-574-Leu（TGG-TTG）.In addition,the ALS sequences of susceptive（MK625097）and resistant（MK625098）L.arvense had been registered in National Center for Biotechnology Information（NCBI）.To the best of our knowledge,this is the first report of ALS gene Trp-574-Leu amino acid mutation confer resistance to tribenuron-methyl in L.arvense.3.Results from in vitro ALS assays indicated that the total ALS activity was similar between the R and S populations.ALS extracted from the R populations with mutation（HNXX-1）was much less susceptible to tribenuron-methyl compared with the S population.The herbicide concentration resulting in 50%reduction of ALS activity（I50）was 117.2-fold greater than the S population.ALS extracted from the R populations without mutation was similar to the S populations.4.The GSTs activity toward CDNB for the untreated and treated plants from each population was analyzed over a period of 6 days.In untreated plants,there wo no significance on the GSTs avtivity for each population during 6 days.The GSTs extracted from the AHXC showed higher GSTs activity than S and other populations during the experimental period.For treated plants,the GSTs activity could be induced by tribenuron-methyl for all tested populations.Especially,the induced GSTs activity of AHXC is greater than S and other populations,and the GSTs activity of AHXC is more rapidly induced by 48h than S and other populations.5.As the cytochrome P450s inhibitor,malathion(1000 g a.i.ha^-1)was used in combination with tribenuron-methyl to evaluate the activity of P450s in R population.The results showed that malathion decrease the GR50 by 92.9%（SDQD-1）and 88.8%（AHXC）for tribenuron-methyl in R population without mutation;malathion decrease the GR₅₀ by 34.9%（HNXX-1）for R population with mutation,8.33-15.4%for S populations,respectively.The results above indicate that non-target resistance caused by P450s may one of the resistance mechanisms in HNXX-1,SDQD-1 and AHXC population.6.Whole-plant dose-response experiments were conducted to determine the cross-resistance of the R populations（HNXX-1,SDQD-1,AHXC）to other herbicides:pyrithiobac-sodium,imazethapyr,florasulam,flucarbazone-sodium,pyroxsulam,carfentrazone-ethyl and fluroxypyr-meptyl.The cross-resistance experiments showed that R populations have evolved various resistance to four ALS-inhibiting herbicides:pyrithiobac-sodium,florasulam,flucarbazone-sodium and pyroxsulam.Results showed that HNXX-1 population was highly resistant to pyrithiobac-sodium,imazethapyr,florasulam,flucarbazone-sodium and pyroxsulam,and the RIs were from 53.0-to 151.2-fold.And SDQD-1 population showed high-level resistance to pyroxsulam with RI of 10.1-fold,middle-level resistance to pyrithiobac-sodium with RI of 9.5-fold,low-level resistance to florasulam with RI of 3.2-fold,susceptibility to imazethapyr.AHXC population showed middle-level resistance to pyrithiobac-sodium with RI of 8.7-fold,low-level resistance to imazethapyr,florasulam,flucarbazone-sodium and pyroxsulam with RIs form 3.0-to 3.8-fold.In addition,all tested L.arvense populations were susceptible to carfentrazone-ethyl and fluroxypyr-meptyl.This study characterized the resistance to tribenuron-methyl in L.arvense 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 Trp574 in the ALS gene.The enhenced herbicide metabolism caused by the enhenced activity of GSTs or P450s maybe associated with the tribenuron-methyl resistance in L.arvense.Cross-resistance experiments indicated that R populations exhibited various resistance patterns to other ALS inhibitors and alternative herbicides.