Preliminary Study On The Target-sited Mechanism Of Penoxsulam Resistance In Echinochloa Crus-galli

Barnyardgrass(Echinochloa crus-galli)is the most important malignant weed in paddy fields in China and even in the world,seriously affecting the yield and the quality of rice.Penoxsulam,which targets on the acetolactate synthase(ALS),was used to control bamyardgrass and other weeds in rice fields since put on the market.Because of its single mode of action,overusing in amount and increase in usage time,the penoxsulam resistance in barnyardgrass begain to appear and developed year by year,causing field control decreased.In 2012,our laboratory collected barnyardgrass populations from different rice regions,and then reported the penoxsulam resistance,but the mechanism of resistance remains unclear.To explore the mechanism of penoxsulam in baryardgrass,we first identified the sensitivity of 32 populations to penoxsulam and detected the level of cross-resistance of five resistant populations to other ALS-inhibitors and multi-resistance to other herbicides of different mode of action,which provides basic information for chemical control.We then explore the mechanism from ALS activity,ALS sequences and ALS gene expression level and preliminary clarify the target-site-based mechanism.The final results were showed as follow.Based on the whole-plant bioassay,the ED50 values of the 32 populations to penoxsulam were determined.According to the standards of this laboratory,of the 32 populations tested,13 populations were sensitive to penoxsulam(relative resistance index?2),and 6 populations showed decreased sensitivity to penoxsulam(2<relative resistance index?4),4 populations showed low resistance to penoxsulam(4<relative resistance index?10),and 5 populations showed moderate resistance to penoxsulam(10<relative resistance index?30).The four populations showed high resistance to penoxsulam(30<relative resistance index?150).All these mean that many baryardgrass populations showed high-level resistance to penoxsulam in some places of China,which consistents with the fact that the control efficiency in the field has decreased.Meanwhile,five populations with different degrees of resistance were selected to study cross-resistance and multi-resistance.For example,highly resistant population AXXZ-2 showed cross-resistance to pyribenzoxim,pyroxsulam,flucetosulfuron,rimsulfuron,metazosulfuron,flucarbazone-sodium,propyrisulfuron and triafamone;highly resistant population AXXZ-6 showed cross-resistance to bispyribac sodium,pyribenzoxim,pyroxsulam,flucetosulfuron,rimsulfuron,metazosulfuron and flucarbazone-sodium;moderately resistant population AXXZ-8 showed cross-resistance to pyroxsulam,flucetosulfuron,flucarbazone-sodium and triafamone;moderately resistant population HYYJ-1 showed cross-resistance to bispyribac sodium,pyribenzoxim,flucetosulfuron,rimsulfuron and metazosulfuron;low-resistance population JNRG-2 showed cross-resistance to metazosulfuron.AXXZ-2,AXXZ-6,AXXZ-8 and HYYJ-1 populations also showed multi-resistance to other herbicides with different modes of action.Still,five resistant populations were sensitive to some soil-treatment herbicides such as oxyfluorfen,oxadiazon,pretilachlor,pendimethalin,mefenacet and one leaf-treatment herbicide propanil,which means these herbicides,can control the resistant populations effectively.Target-site mechanism includes ALS activity,ALS gene sequences and ALS gene expression level.It's raveled that the total ALS activity was very closed between six populations without penoxsulam.While treated with penoxsulam,AXXZ-2,AXXZ-6,AXXZ-8,JNRG-2 and HYYJ-1 showed different degree decrease in activity compared to the JLGY-3 and the RIs were 8.02,8.43,6.00,4.86 and 3.07 respectively calculated with IC50 values,which suggested that an insensitive of ALS activity was the cause for the resistance to penoxsulam in resistant populations.Using PCR,we got complete ALS gene sequences of six populations,and the sequences have 2300bp approximately with slightly difference among populations.The coding region is about 1930bp and encodes about 643 amino acids.All the same is that there is no intron area.What is interesting is that the ALS gene copies number varied(AXXZ-2,AXXZ-6 and AXXZ-8 with 3 copies,while JNRG-2,HYYJ-1 and JLGY-3 with 2 copies).Compared to the ALS gene of sensitive population,an Ala-205-Val substation was found in AXXZ-2 population,a Trp-574-Arg mutant was detected in AXXZ-6 population and an Ala-122-Gly mutant in JNRG-2 population was found.Three reported target-site mutants might be responsible for the resistance in these populations,while no known ALS mutant was detected in HYYJ-1 and AXXZ-8 population and the mechanism remains to be clarified.By the qPCR method,the expression levels of ALS gene were measured in six populations.The results revealed that the expression levels increased to the top value at the third day after the penoxsulam treatment in all populations and the expression levels of JLGY-3 and JNRG-2 populations were higher than other resistant population.We speculated that the inherent difference among populations maybe the cause and the expression level up-regulation could not be the reason for the penoxsulam resistance in these populations.In conclusion,we firstly determined the sensitivity of 32 populations to penoxsulam and then characterized the pattern of cross-resistance and multi-resistance of 5 selected resistant populations,defining the sensitivity of 5 resistant populations to common herbicides.We studied the target-sited basis of penoxsulam resistance using 5 resistant populations as materials.It's clarified that the three reported amino acid mutations of target enzyme ALS and elevated ALS activity are important causes of penoxsulam resistance,and the difference of ALS expression is not responsible for the resistance.