Study On Metabolic Resistance To Bensulfuron-Methyl In Threeleaf Arrowhead(Sagittaria Trifolia L.)

Threeleaf arrowhead(Sagittaria trifolia L)is a common weed in rice crop,which is widely distributed in the northeastern three provinces region in China.Bensufulron-methyl is a sulfonylureas herbicide that is commonly used to control broadleaf weeds in rice fields in China and it has been used for more than 30 years.In early years,bensulfuron-methyl had a good control effect on S.trifolia,but its control effect was significantly decreased in the past 10 years,resulting in reduced rice growth and yield.In order to abundantly characterize the resistance level and identify the resistance mechanisms to bensulfuron-methyl in S.trifolia of rice fields collected from the northeastern regions in China,and provide a preliminary theoretical reference for the effective control of resistant S.trifolia.The objectives of this study were: to determine the levels of resistance to bensulfuron-methyl in 38 S.trifolia populations that were collected from Liaoning Jilin and Heilongjiang provinces;to identify suspected metabolic resistance S.trifolia population by P450 inhibitors(malathion and piperonyl butoxide);to determine the levels of resistance to different classes of ALS-inhibitors with or without malathion in S.trifolia populations;to analyze differences of bensulfuron-methyl absorption and metabolism in metabolic resistant and sensitive S.trifolia populations by HPLC-MS/MS;to identifiy genes involved in metabolic resistance to bensulfuron-methyl in resistant S.trifolia population by RNA-Seq.The main results of this study are as follows:1.A single dose-response experiment was used to identify the sensitivity to bensulfuronmethyl in all the 38 S.trifolia populations.The application rate for treatment were maximum field recommended dose(45 g a.i./hm2).It was found that all the 36 S.trifolia populations collected from the rice fields were resistant to bensulfuron-methyl,S1 and S2 collected from Hunhe and Longtan lake in Shenyang were sensitive populations.The resistance levels of all the S.trifolia populations to bensulfuron-methyl were determined;the results showed that 36 resistant S.trifolia populations exhibited different levels of resistance to bensulfuron-methyl,and the resistance index(RI)was arranging from 14.88 to 91.90;the resistance level of J-3 that was collected from Yushu city in Jilin province was the highest in all selected populations.Malathion and piperonyl butoxide(P450 inhibitors)displayed different synergistic effects on bensulfuron-methyl,but showed significant synergistic to population of Liaoyang city in Liaoning province(L-9)and Shulan city Jilin province(J-1),leading the inhibition rate of bensulfuron-methyl on L-9 and J-1 populations to increase from 35.9% to 75.4% and 54.1% to 63.4%,respectively.2.Through L-9 population the single plant isolation and purification,a subpopulation BC1 without ALS gene mutation was derived.The levels of resistance to five ALS-inhibiting herbicides(bensulfuron-methyl,pyrazosulfuron-methyl,ethoxysulfuron,penoxsulam and bispyribac-sodium)were determined,and whole-plant dose-response experiments showed that the BC1 population exhibited cross resistance to five ALS herbicides,resistance ratios was ranging from 2.14 to 51.53.The total ALS activity and susceptibility in vitro to bensulfuronmethyl between S1 and BC1 populations was not significant difference,and the results indicated that the resistance to ALS-inhibitors in BC1 population was not correlated with its ALS activity.The effects of malathion on the resistance of BC1 and S1 populations were investigated,and results showed that the resistance levels to bensulfuron-methyl in BC1 population could be significantly reversed with 1.8~6.9 fold by malathion,indicating that P450 activity of BC1 was higher than S1 population.In summary,the results of the present study demonstrated that the BC1 population has evolved metabolic resistance to bensulfuron-methyl mediated by P450 s.3.The absorption and metabolism differences of bensulfuron-methyl in BC1 and S1 populations was conducted by HPLC-MS/MS.The results indicated that absorption of bensulfuron-methyl by BC1 and S1 populations showed no significant differences at 0.5,1,3,5,7 and 9 days after treatment;and the BC1 population metabolized bensulfuron-methyl significantly faster than S1 population at 0.5 days after treatment.After treated by malathion,the bensulfuron-methyl absorption in BC1 and S1 populations were significantly enhanced,but there were no significant difference between them.Malathion could significantly inhibited the metabolism of bensulfuron-methyl in BC1 population,but not in S1 population.The results indicated that the resistance to bensulfuron-methyl in BC1 and S1 populations was not related to its absorptive capacity,but was closely related to the difference in the metabolic capacity to bensulfuron-methyl.4.RNA-Seq was conducted to identifiy genes involved in metabolic resistance to bensulfuron-methyl in S.trifolia populations.Total 15 up-regulated genes related to metabolism were identified,including 4 glutathione-S-transferases(GSTs),5 cytochrome P450 monooxygenases(P450s),1 ABC transporters and 5 Peroxidase.Further verification by realtime PCR showed that 1 GSTs,2 P450 s,1 Peroxidase and 1 ABC transporters were significantly expressed in the resistant BC1 population.It was concluded that the up-regulation of metabolic related genes resulted in metabolic resistance to bensulfuron-methyl in S.trifolia population.